Transgenic mouse comprising a polynucleotide encoding human or humanized C5aR and methods of production and use

Abstract

The present invention relates to transgenic non-human mammals comprising a polynucleotide encoding a human or humanized C5aR. The invention also relates to use of the transgenic non-human mammals in methods of screening for agonists, inverse agonists and antagonists of human C5aR and for testing efficacy of C5aR agonists, inverse agonists and antagonists in various animal models of disease.

1. A transgenic mouse comprising a polynucleotide encoding a human C5aR or humanized C5aR, wherein the C5a endogenous to the mouse binds to and effects signalling of the human or humanized C5aR, wherein said signalling is capable of inducing arthritis upon administration of sera from arthritic K/BxN mice, and wherein the transgenic mouse is homozygous for the polynucleotide encoding a human or humanized C5aR and wherein the endogenous C5aR coding sequences are disrupted. 
     
     
       2. The transgenic mouse according to  claim 1 , wherein the polynucleotide encodes human C5aR comprising the amino acid sequence as shown in SEQ ID NO:3, or an allelic variant thereof. 
     
     
       3. The transgenic mouse according to  claim 1 , wherein the polynucleotide comprises the nucleotide sequence as shown in SEQ ID NO:2, or an allelic variant thereof. 
     
     
       4. The transgenic mouse according to  claim 1 , wherein the polynucleotide encodes humanized C5aR. 
     
     
       5. The transgenic mouse according to  claim 4 , wherein the humanized C5aR comprises a C5aR sequence endogenous to the mouse wherein at least one extracellular or intracellular domain is replaced with the corresponding human C5aR domain. 
     
     
       6. The transgenic mouse according to  claim 1 , wherein the endogenous C5aR coding sequences have been replaced with a corresponding human C5aR coding sequence. 
     
     
       7. An isolated cell(s), cell line, tissue or organ obtained from the transgenic mouse of  claim 1 , the isolated cell, cell line, tissue or organ comprising a polynucleotide encoding a human C5aR or humanized C5aR. 
     
     
       8. A method for producing a transgenic mouse for testing compounds for an effect on a phenotype associated with C5aR signalling, the method comprising:
 introducing into the genome of a mouse a polynucleotide construct encoding human C5aR, humanized C5aR or a fragment of human C5aR to produce a transgenic mouse, wherein the C5a endogenous to the mouse binds to and effects signalling of the human or humanized C5aR, wherein said signalling is capable of inducing arthritis upon administration of sera from arthritic K/BxN mice, and wherein the endogenous C5aR coding sequences are disrupted. 
 
     
     
       9. The method according to  claim 8 , wherein the polynucleotide construct encodes human C5aR. 
     
     
       10. The method according to  claim 9 , wherein the polynucleotide construct encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:3, or an allelic variant thereof. 
     
     
       11. The method according to  claim 9 , wherein the polynucleotide construct comprises the nucleotide sequence as shown in SEQ ID NO:2, or an allelic variant thereof. 
     
     
       12. The method according to  claim 8 , wherein the polynucleotide construct encodes humanized C5aR. 
     
     
       13. The method according to  claim 8 , wherein the polynucleotide construct encodes a fragment of human C5aR. 
     
     
       14. The method according to  claim 13 , wherein the fragment encompasses at least one extracellular domain of human C5aR. 
     
     
       15. The method according to  claim 8 , wherein the method comprises replacing the endogenous C5aR coding sequences, with a corresponding human C5aR coding sequence or fragment thereof. 
     
     
       16. A method for screening a candidate compound for anti-inflammatory activity in the transgenic mouse according to  claim 1 , or isolated tissue or cells obtained therefrom, the method comprising:
 administering a candidate compound to the transgenic mouse, wherein an inflammatory response is induced in the transgenic mouse by administration of sera from arthritic K/BxN mice; and 
 examining an of the candidate compound on the inflammatory response in the transgenic mouse or isolated tissue or cells obtained therefrom; 
 wherein a decrease in the inflammatory response in the transgenic mouse, or isolated tissue or cells obtained therefrom, as compared to the inflammatory response in the absence of the candidate compound, indicates the candidate compound has anti-inflammatory activity. 
 
     
     
       17. The method according to  claim 16  wherein the candidate compound is selected from the group consisting of: a peptide, including a peptide derived from C5aR or C5a or other non-C5aR peptide and capable of inhibiting, reducing or repressing a C5aR function, a C5aR dominant-negative mutant; a non peptide inhibitor of C5aR; an antibody or antibody fragment which binds to C5aR and inhibits a C5aR function; a small organic molecule, a nucleic acid encoding said peptide derived from C5aR or C5a or other non-C5aR peptide inhibitor, an antisense nucleic acid directed against C5aR-encoding mRNA, an anti-C5aR ribozyme, and a small interfering RNA (RNAi) that targets C5aR gene expression.